Methods for generating immunity to antigen

ABSTRACT

Provided are methods of generating an immune response to an antigen. The method comprises priming an individual by administering an expression vector encoding the antigen. The vector comprises a transcription unit encoding a secretable fusion protein, the fusion protein containing an antigen and CD40 ligand. Administration of a fusion protein containing the antigen and CD40 ligand is used to enhance the immune response above that obtained by vector administration alone. The methods may be used to generate an immune response against cancer expressing a tumor antigen such as a mucin or human papilloma viral tumor antigen and to generate an immune response against an infectious agent. Also provided is a method for simultaneously producing the expression vector and the fusion protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/009,533 filed on Dec. 10, 2004, which claims priority to U.S.Provisional Patent Application Ser. No. 60/529,016 filed on Dec. 11,2003, the disclosures of which including the figures are each herebyincorporated herein by reference.

GOVERNMENTAL RIGHTS

This invention was mae with Government support under Contract NumbersDAMD17-03-1-0554 and DAMD17-99-1-9457 funded by the U.S. Army CancerCenter, and under funding agreement R43 CA108051 funded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to methods of developing immunity againstan antigen using an expression vector that expresses a secretable fusionprotein comprising an antigen fused to CD40 ligand. The methods alsorelate to an immunization scheme of priming with the expression vectorand boosting with a protein antigen. The invention also relates to anapproach for producing the vector and the protein antigen simultaneouslyin a production cell system.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.This application claims priority to U.S. application Ser. No.60/529,016, filed Dec. 11, 2003, which is incorporated herein in itsentirety including the drawings. Applications related to thisapplication are PCT/US03/36237 filed Nov. 12, 2003 entitled “adenoviralvector vaccine” and U.S. provisional patent applications 60/524,925(filed Nov. 24, 2003), 60/525,552 (filed Nov. 25, 2003), and 60/529,015(filed Dec. 11, 2003), all of which are incorporated herein in theirentirety including the drawings.

The activation of antigen presenting cells (APCs) which includes thedendritic cells (DCs), followed by loading of the antigen presentingcell with relevant antigens, is a requisite step in the generation of aT cell dependent immune response against cancer cells. Once activatedand loaded with tumor antigens, DCs migrate to regional lymph nodes(LNs) to present antigens to T cells. Very commonly, these APCs expressinsufficient amounts of surface activation molecules which are requiredfor optimal activation and expansion of T cell clones competent torecognize tumor antigens. See Shortman, et al., Stem Cells 15:409-419,1997.

Antigen presentation to naive T cells, in the absence of costimulatorymolecule expression on the surface of the APC, leads to anergy of the Tcells. See Steinbrink, et al. Blood 99: 2468-2476, 2002. Moreover,cross-presentation by DCs without CD4⁺ T cell help also results inperipheral deletion of Ag-specific T cells in regional LNs. SeeKusuhara, et al., Eur J Immunol 32:1035-1043, 2002. In contrast, in thepresence of CD4⁺ T cell help, DCs acquire functional ability tocross-prime T cells, resulting in clonal expansion of effector T cells.See Gunzer, et al., Semin Immunol 13:291-302, 2001. This CD4⁺ T cellhelp can be replaced with CD4O-CD40 ligand (CD40L) interactions. SeeLuft, et al. Int Immunol 14:367-380, 2002. CD40L is a 33-kDa type IImembrane protein and a member of the TNF gene family and is transientlyexpressed on CD4⁺ T cells after TCR engagement. See Skov, et al. JImmunol. 164: 3500-3505, 2000.

The ability of DCs to generate anti-tumor immune responses in vivo hasbeen documented in a number of animal tumor models. See Paglia, et al. JExp Med 183: 317-322, 1996; Zitvogel, et al., J Exp Med. 183: 87-97,1996. However, DC-mediated induction of immunity represents a majortherapeutic challenge. It is considered difficult to ensure that theantigen presenting cells express appropriate adhesion molecules andchemokine receptors to attract DCs to secondary lymphoid organs forpriming T cells. See Fong, et al. J Immunol. 166: 4254-4259, 2001;Markowicz, et al. J Clin Invest. 85: 955-961, 1990; Hsu, et al. Nat Med.2: 52-58, 1996; Nestle, et al. Nat Med. 4: 328-332, 1998; Murphy, etal., Prostate 38: 73-78, 1999; Dhodapkar, et al. J Clin Invest. 104:173-180, 1999.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to the use of a fusion proteinin developing an immune response to an antigen. In a preferredembodiment, an immune response to an antigen is obtained byadministering an expression vector encoding a secretable fusion protein.The vector includes a transcription unit encoding a secretable fusionprotein which contains the antigen and CD40 ligand. The fusion proteinis also administered before, concurrently or after administration of thevector. Preferably, the fusion protein is administered after the vector.

In one approach, the sequence encoding the antigen in the fusion proteintranscription unit is 5′ to sequence encoding the CD40 ligand. Inanother approach, the sequence encoding the CD40 ligand in the fusionprotein transcription unit is 5′ to sequence encoding the antigen. In apreferred embodiment, the CD40 ligand lacks all or a portion of itstransmembrane domain

The antigen may be any antigen to which an immune response may begenerated in an individual. In preferred embodiments, the antigen is atumor antigen; the tumor antigen is the E6 or E7 protein of humanpapilloma virus; the tumor antigen is a mucin antigen, which may beselected from the group consisting of MUC1, MUC2, MUC3A, MUC3B, MUC4,MUCSAC, MUCSB, MUC6, MUC7, MUC8, MUC9, MUC12, MUC13, MUC15, and MUC16;the mucin antigen is from MUC1; the human epidermal growth factor (EGF)like receptor (e.g., HER1, HER2, HERS and HER4), the antigen is aninfectious agent antigen; the infectious agent antigen is a viralantigen; the infectious agent viral antigen is from human papillomavirus; the viral antigen is the E6 or E7 protein of human papillomavirus.

In another aspect, the invention provides methods of treating anindividual with cancer that expresses a tumor antigen. The methodincludes administering the expression vector which includes atranscription unit encoding a secretable fusion protein that containsthe tumor antigen and CD40 ligand. The fusion protein is alsoadministered before, concurrently or after administration of the vector.Preferably, the fusion protein is administered after the vector.

In a further aspect, the invention provides a method of generatingimmunity in a subject to an infectious agent. The method includesadministering the expression vector which includes a transcription unitencoding a secretable fusion protein that contains the infectious agentantigen and CD40 ligand. The fusion protein is also administered before,concurrently or after administration of the vector. Preferably, thefusion protein is administered after the vector.

In yet a further aspect, the invention relates to an approach forproducing the vector and the fusion protein together in the same hostproduction cell system. In a preferred embodiment, the fusion protein isexpressed from the same vector used to generate immunity by vaccination.In this way, both the vector and the fusion protein can be producedsimultaneously through a single production system.

In preferred embodiments, the expression vector may be a viralexpression vector or a non-viral expression vector; the expressionvector may be an adenoviral vector; the vector may be advantageouslyadministered subcutaneously; the vector may be administered on asubsequent occasion(s) to increase the immune response; a signalsequence may be placed upstream of the fusion protein for secretion ofthe fusion protein; immunity against the antigen may be long lasting andinvolve generation of cytotoxic CD8⁺ T cells against antigen expressingcells and the production of antibody to the antigen; the transcriptionunit may include sequence that encodes a linker between the antigen andthe CD40 ligand; suitable linkers may vary in length and composition;the expression vector may include a human cytomegaloviruspromoter/enhancer for controlling transcription of the transcriptionunit; and the CD40 ligand may be a human CD40 ligand.

Abbreviations used herein include “Ad” (adenoviral); “sig” (signalsequence); and “ecd” (extracellular domain).

These and other embodiments are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence encoding human MUC1 (SEQ ID NO:1)

FIG. 2 shows the amino acid sequence of human MUC1 (SEQ ID NO:2).

FIG. 3 shows the level of interferon gamma produced in an ELISA spotassay using spleen cells from MUC-1 transgenic animals (hMUC-1.Tg)primed with adenoviral expression vector Ad-K/ecdhMUC1-ΔCtΔTmCD40L andboosted subcutaneously with either expression vector or the maturefusion protein ecdhMUC1-ΔCtΔTmCD40L. The various treatment groupsinclude protein boost seven days after two weekly vector injections(T1), two weeks after two weekly vector injections (T2), one week afterone vector injection (T3), and two weeks after one vector injection(T4). In T5, two subcutaneous protein injections (administered two weeksapart) were given starting 7 days after a single vector injection. InControl, two vector injections were given without protein.

FIG. 4 shows the level of T cell cytotoxicity from MUC-1 transgenicanimals (hMUC-1.Tg) primed with adenoviral expression vectorAd-K/ecdhMUC1-ΔCtΔTmCD40L and boosted subcutaneously with eitherexpression vector or the mature fusion protein ecdhMUC1-ΔCtΔTmCD40L. Thevarious treatment groups are as described in FIG. 3.

FIG. 5 shows the level of antibody against fusion proteinecdhMUC1-ΔCtΔTmCD40L in serum of MUC-1 transgenic animals (hMUC-1.Tg)primed with adenoviral expression vector Ad-K/ecdhMUC1-ΔCtΔTmCD40L andboosted subcutaneously with either expression vector or the maturefusion protein ecdhMUC1-ΔCtΔTmCD40L. The various treatment groups are asdescribed in FIG. 3. Antibodies were detected in an ELISA. Microwellplates coated with the fusion protein ecdhMUC1-ΔCtΔTmCD40L were reactedwith serum, washed and bound mouse antibody detected using ratanti-mouse antibody conjugated to horseradish peroxidase.

FIG. 6 shows the level growth of MUC1 expressing tumor cells(LL2/LL2hMUC-1) in MUC-1 transgenic animals (hMUC-1.Tg) administeredadenoviral expression vector Ad-K/ecdhMUC1-ΔCtΔTmCD40L versus one or twosubsequent administrations of fusion protein ecdhMUC1-ΔCtΔTmCD40L.

FIG. 7 demonstrates tumor prevention in animals immunized withAd-sig-ecdhMUC-1/ΔCtΔTmCD40L vector and ecdhMUC-1/ΔCtΔTm CD40L protein.VVV=three Ad-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionsadministered on days 1, 7 and 21; PPP=three ecdhMUC-1/ΔCtΔTm CD40Lprotein subcutaneous injections administered on days 1, 7 and 21; orVPP=a single Ad-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionfollowed at days 7 and 21 by ecdhMUC-1/ΔCtΔTm CD40L protein subcutaneousinjections. One week later (day 28), mice were injected subcutaneouslywith five hundred thousand LL2/LL1hMUC-1 lung cancer cells. Two weekslater (day 42), 500,000 of the LL2/LL1hMUC-1 tumor cells wereadministered intravenously to test mice via the tail vein. Multipleadministrations of vector alone or vector followed by boosting withprotein was effective in preventing the establishment of human tumors inmice.

FIG. 8 demonstrates the levels of hMUC-1 specific antibodies invaccinated test mice at 63 days following the start of the vaccination.VVV=three Ad-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionsadministered on days 1, 7 and 21; PPP=three ecdhMUC-1/ΔCtΔTm CD40Lprotein subcutaneous injections administered on days 1, 7 and 21; orVPP=a single Ad-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionfollowed at days 7 and 21 by ecdhMUC-1/ΔCtΔTm CD40L protein subcutaneousinjections. The schedule of one Ad-sig-ecdhMUC-1/deltaCtdeltaTmCD40Lvector subcutaneous injection followed by two successiveecdhMUC-1/deltaCtdeltaTmCD40L protein subcutaneous injections at 7 and21 days following the vector injection induced the highest levels ofhMUC-1 specific antibodies.

FIG. 9 demonstrates subcutaneous tumor therapy (post establishment) inanimals immunized with Ad-sig-ecdhMUC-1/ΔCtΔTmCD40L vector andecdhMUC-1/ΔCtΔTm CD40L protein. VVV=three Ad-sig-ecdhMUC-1/ΔCtΔTm CD40Lvector subcutaneous injections administered on days 5, 12 and 26;PPP=three ecdhMUC-1/ΔCtΔTm CD40L protein subcutaneous injectionsadministered on days 5, 12 and 26; or VPP=a singleAd-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injection followed atdays 12 and 26 by ecdhMUC-1/ΔCtΔTm CD40L protein subcutaneousinjections. Subcutaneous tumor (500,000 of the LL2/LL1hMUC-1) wasadministered on day 1 and vaccinations were carried out at day 5. Tumorwas administered i.v. on day 40 and tumor development (subcutaneous andlung) evaluated at day 54.

FIG. 10 demonstrates lung metastatic tumor nodule therapy (postestablishment) in the animals treated as described in FIG. 9. Leftpanel: The results were similar to the subcutaneous tumor preventionwith schedule VVV and VPP most effective. Right panel: the combinationof one vector injection followed by two protein injections (VPP)completely suppressed the growth of established lung nodules of thehMUC-1 positive cancer cells.

FIG. 11 compares various boosting strategies following a singlesubcutaneous administration of Ad-sig-ecdhMUC-1/ecdCD40L vector on theability of animals to resist development of a MUC-1 expressing tumor.ecdhMUC-1/ecdCD40L protein in bacterial extract; ecdhMUC-1 linked to thekeyhole limpet hemocyaninin (KLH), with or without incomplete Freund'sadjuvant; PBS (phosphate buffered saline); and control bacterial extract(bacterial host strain not infected with Ad-sig-ecdhMUC-1/ecdCD40Lvector.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, a method is provided forgenerating an immune response against an antigen using an expressionvector. The vector includes a transcription unit encoding a secretablefusion protein containing an antigen and CD40 ligand. In a preferredembodiment, the transcription unit includes from the amino terminus, asecretory signal sequence, an antigen, a linker and a secretable form ofCD40 ligand. In preferred embodiments, the secretable form of CD40ligand lacks all or substantially all of its transmembrane domain

In a preferred approach, the individual is first administered the vectoron one or more occasions to generate a primary immune response. Thefusion protein is also administered in an effective amount afteradministration of vector to boost the immune response to the antigenabove that obtained with vector administration alone.

The term “in an effective amount” in reference to administering thefusion protein is an amount that generates an increased immune responseover that obtained using the expression vector alone. A time intervalbetween administrations is generally required for optimal results. Anincrease in the immune response may be measured as an increase in T cellactivity or antibody production (see e.g., FIGS. 3-5). Generally, atleast one week between vector administration and protein boosting iseffective although a shorter interval may be possible. An effectivespacing between administrations may be from 1 week to 12 weeks or evenlonger. Multiple boosts may be given which may be separated by from 1-12weeks or even longer periods of time.

The use of the fusion protein to boost the immune response avoids havingto repetitively administer the expression vector which might generatehypersensitivity to multiple injections. The antigen portion of thefusion protein is preferably the fusion protein which is encoded by thetranscription unit of the expression vector used in the initialadministration. However, the antigen portion of the fusion protein maydiffer from the encoded antigen provided that there is at least oneshared antigenic determinant or epitope common to the antigen of theexpression vector and that of the fusion protein used for boosting.

The fusion protein may be prepared in a mammalian cell line system,which is complementary to the vector. Example in the case of adenovirus,the cell line system can be 293 cells that contain the Early Region 1(E1) gene and can support the propagation of the E1-substitutedrecombinant adenoviruses. When the adenoviral vectors infect theproduction cells, the viral vectors will propagate themselves followingthe viral replication cycles. However, the gene of interest that iscarried by the viral vector in the expression cassette will expressduring the viral propagation process. This can be utilized forpreparation of the fusion protein encoded by the vector in the samesystem for production of the vector. The production of both the vectorand the fusion protein will take place simultaneously in the productionsystem. The vector and protein thus produced can be further isolated andpurified via different processes.

The fusion protein may be administered parenterally, such asintravascularly, intravenously, intraarterially, intramuscularly,subcutaneously, or the like. Administration can also be orally, nasally,rectally, transdermally or inhalationally via an aerosol. The proteinboost may be administered as a bolus, or slowly infused. The proteinboost is preferably administered subcutaneously.

The fusion protein boost may be formulated with an adjuvant to enhancethe resulting immune response. As used herein, the term “adjuvant” meansa chemical that, when administered with the vaccine, enhances the immuneresponse to the vaccine. An adjuvant is distinguished from a carrierprotein in that the adjuvant is not chemically coupled to the immunogenor the antigen. Adjuvants are well known in the art and include, forexample, mineral oil emulsions (U.S. Pat. No. 4,608,251, supra) such asFreund's complete or Freund's incomplete adjuvant (Freund, Adv. Tuberc.Res. 7:130 (1956); Calbiochem, San Diego Calif.), aluminum salts,especially aluminum hydroxide or ALLOHYDROGEL (approved for use inhumans by the U.S. Food and Drug Administration), muramyl dipeptide(MDP) and its analogs such as [Thr¹]-MDP (Byers and Allison, Vaccine5:223 (1987)), monophosphoryl lipid A (Johnson et al., Rev. Infect. Dis.9:S512 (1987)), and the like.

The fusion protein can be administered in a microencapsulated or amacroencapsulated form using methods well known in the art. Fusionprotein can be encapsulated, for example, into liposomes (see, forexample, Garcon and Six, J. Immunol. 146:3697 (1991)), into the innercapsid protein of bovine rotavirus (Redmond et al., Mol. Immunol. 28:269(1991)) into immune stimulating molecules (ISCOMS) composed of saponinssuch as Quil A (Morein et al., Nature 308:457 (1984)); Morein et al., inImmunological Adjuvants and Vaccines (G. Gregoriadis al. eds.)pp.153-162, Plenum Press, NY (1987)) or into controlled-releasebiodegradable microspheres composed, for example, of lactide-glycolidecompolymers (O′Hagan et al., Immunology 73:239 (1991); O′Hagan et al.,Vaccine 11:149 (1993)).

The fusion protein also can be adsorbed to the surface of lipidmicrospheres containing squalene or squalane emulsions prepared with aPLURONIC block-copolymer such as L-121 and stabilized with a detergentsuch as TWEEN 80 (see Allison and Byers, Vaccines: New Approaches toImmunological Problems (R. Ellis ed.) pp. 431-449, Butterworth-Hinemann,Stoneman N.Y. (1992)). A microencapsulated or a macroencapsulated fusionprotein can also include an adjuvant.

The fusion protein also may be conjugated to a carrier or foreignmolecule such as a carrier protein that is foreign to the individual tobe administered the protein boost. Foreign proteins that activate theimmune response and can be conjugated to a fusion protein as describedherein include proteins or other molecules with molecular weights of atleast about 20,000 Daltons, preferably at least about 40,000 Daltons andmore preferably at least about 60,000 Daltons. Carrier proteins usefulin the present invention include, for example, GST, hemocyanins such asfrom the keyhole limpet, serum albumin or cationized serum albumin,thyroglobulin, ovalbumin, various toxoid proteins such a tetanus toxoidor diptheria toxoid, immunoglobulins, heat shock proteins, and the like.

Methods to chemically couple one protein to another (carrier) proteinare well known in the art and include, for example, conjugation by awater soluble carbodiimide such as1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride, conjugationby a homobifunctional cross-linker having, for example, NHS ester groupsor sulfo-NHS ester analogs, conjugation by a heterobifunctionalcross-linker having, for example, and NHS ester and a maleimide groupsuch as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate and, conjugation with gluteraldehyde (see, forexample, Hermanson, Bioconjugate Techniques, Academic Press, San Diego,Calif. (1996)); see, also, U.S. Pat. Nos. 4,608,251 and 4,161,519).

The term “vector” which contains a transcription unit (aka. “expressionvector”) as used herein refers to viral and non-viral expression vectorsthat when administered in vivo can enter target cells and express anencoded protein. Viral vectors suitable for delivery in vivo andexpression of an exogenous protein are well known and include adenoviralvectors, adeno-associated viral vectors, retroviral vectors, herpessimplex viral vectors, and the like. Viral vectors are preferably madereplication defective in normal cells. See U.S. Pat. Nos. 6,669,942;6,566,128; 6,794,188; 6,110, 744; 6,133,029.

As used herein, the term “cells” is used expansively to encompass anyliving cells such as mammalian cells, plant cells, eukaryotic cells,prokaryotic cells, and the like.

The term “adenoviral expression vector” as used herein, refers to anyvector from an adenovirus that includes exogenous DNA inserted into itsgenome which encodes a polypeptide. The vector must be capable ofreplicating and being packaged when any deficient essential genes areprovided in trans. An adenoviral vector desirably contains at least aportion of each terminal repeat required to support the replication ofthe viral DNA, preferably at least about 90% of the full ITR sequence,and the DNA required to encapsidate the genome into a viral capsid. Manysuitable adenoviral vectors have been described in the art. See U.S.Pat. Nos. 6,440,944 and 6,040,174 (replication defective E1 deletedvectors and specialized packaging cell lines). A preferred adenoviralexpression vector is one that is replication defective in normal cells.

Adeno-associated viruses represent a class of small, single-stranded DNAviruses that can insert their genetic material at a specific site onchromosome 19. The preparation and use of adeno-associated viral vectorsfor gene delivery is described in U.S. Pat. No. 5,658,785.

Non-viral vectors for gene delivery comprise various types of expressionvectors (e.g., plasmids) which are combined with lipids, proteins andother molecules (or combinations of thereof) in order to protect the DNAof the vector during delivery. Fusigenic non-viral particles can beconstructed by combining viral fusion proteins with expression vectorsas described. Kaneda, Curr Drug Targets (2003) 4(8):599-602.Reconstituted HVJ (hemagglutinating virus of Japan; Sendaivirus)-liposomes can be used to deliver expression vectors or thevectors may be incorporated directly into inactivated HVJ particleswithout liposomes. See Kaneda, Curr Drug Targets (2003) 4(8):599-602.DMRIE/DOPE lipid mixture are useful a vehicle for non-viral expressionvectors. See U.S. 6,147,055. Polycation-DNA complexes also may be usedas a non-viral gene delivery vehicle. See Thomas et al., Appl MicrobiolBiotechnol (2003) 62(1):27-34.

The term “transcription unit” as it is used herein in connection with anexpression vector means a stretch of DNA that is transcribed as asingle, continuous mRNA strand by RNA polymerase, and includes thesignals for initiation and termination of transcription. For example, inone embodiment, a transcription unit of the invention includes nucleicacid that encodes from 5′ to 3,′ a secretory signal sequence, an antigenand CD40 ligand. The transcription unit is in operable linkage withtranscriptional and/or translational expression control elements such asa promoter and optionally any upstream or downstream enhancerelement(s). A useful promoter/enhancer is the cytomegalovirus (CMV)immediate-early promoter/enhancer. See U.S. Pat. Nos. 5,849,522 and6,218,140.

The term “secretory signal sequence” (aka. “signal sequence,” “signalpeptide,” leader sequence,” or leader peptide“) as used herein refers toa short peptide sequence, generally hydrophobic in charter, includingabout 20 to 30 amino acids which is synthesized at the N-terminus of apolypeptide and directs the polypeptide to the endoplasmic reticulum.The secretory signal sequence is generally cleaved upon translocation ofthe polypeptide into the endoplasmic reticulum. Eukaryotic secretorysignal sequences are preferred for directing secretion of the exogenousgene product of the expression vector. A variety of suitable suchsequences are well known in the art and include the secretory signalsequence of human growth hormone, immunoglobulin kappa chain, and thelike. In some embodiments the endogenous tumor antigen signal sequencealso may be used to direct secretion.

The term “antigen” as used herein refers broadly to any antigen to whichan individual can generate an immune response. “Antigen” as used hereinrefers broadly to molecule that contains at least one antigenicdeterminant to which the immune response may be directed. The immuneresponse may be cell mediated or humoral or both.

As is well known in the art, an antigen may be protein in nature,carbohydrate in nature, lipid in nature, or nucleic acid in nature, orcombinations of these biomolecules. An antigen may include non-naturalmolecules such as polymers and the like. Antigens include self antigensand foreign antigens such as antigens produced by another animal orantigens from an infectious agent. Infectious agent antigens may bebacterial, viral, fungal, protozoan, and the like.

The term “tumor associated antigen” (TAA) as used herein refers to aprotein which is present on tumor cells, and on normal cells duringfetal life (onco-fetal antigen), after birth in selected organs, or onmany normal cells, but at much lower concentration than on tumor cells.A variety of TAA have been described. An exemplary TAA is a mucin suchas MUC1, described in further detail below or the HER2 (neu) antigenalso described below. In contrast, tumor specific antigen (TSA) (aka.“tumor-specific transplantation antigen or TSTA) refers to a proteinabsent from normal cells. TSAs usually appear when an infecting virushas caused the cell to become immortal and to express a viralantigen(s).

An exemplary viral TSA is the E6 or E7 proteins of HPV type 16. TSAs notinduced by viruses include idiotypes the immunoglobulin idiotypesassociated with \B cell lymphomas or the T cell receptor (TCR) on T celllymphomas.

An exemplary viral TSA is the E6 or E7 proteins of HPV type 16. HPV cancause a variety of epithelial lesions of the skin and genital tract. HPVrelated diseases of the genital tract constitute the second leadingcause of cancer death among women in the world. These include genitalwarts, cervical intraepithelial neoplasia (CIN) and cancer of thecervix. The HPV type most commonly associated with high grade CIN andcervical cancer is HPV type 16. The majority of cervical cancers expressthe non-structural HPV16-derived gene products E6 and E7 oncoproteins.In HPV-induced cervical cancer model, the E6/E7 oncoproteins arerequired for maintenance of the malignant phenotype and their expressioncorrelates with the transforming potential of HPV16. In addition tousing E6 or E7 as the tumor antigen, one may use an antigenic fragmentof these proteins instead. An antigenic fragment may be determined bytesting the immune response with portions of the molecule such as arepredicted to carry an epitope using well known computer algorithms (e.g.Hopp and Woods hydrophobicity analysis).

TSAs not induced by viruses can be idiotypes of the immunoglobulin on Bcell lymphomas or the T cell receptor (TCR) on T cell lymphomas.Tumor-associated antigens (TAA) are more common than TSA.

Both TAA and TSA may be the immunological target of an expression vectorvaccine. Unless indicated otherwise, the term “tumor antigen” is usedherein to refer collectively to TAA and TSA.

The term “mucin ” as used herein refers to any of a class of highmolecular weight glycoproteins with a high content of clusteredoligosaccharides O-glycosidically linked to tandem repeating peptidesequences which are rich in threonine, serine and proline. Mucin plays arole in cellular protection and, with many sugars exposed on theextended structure, effects multiple interactions with various celltypes including leukocytes and infectious agents. Mucin antigens alsoinclude those identified as CD227, Tumor-associated epithelial membraneantigen (EMA), Polymorphic epithelial mucin (PEM), Peanut-reactiveurinary mucin (PUM), episialin, Breast carcinoma-associated antigen DF3,H23 antigen, mucin 1, Episialin, Tumor-associated mucin,Carcinoma-associated mucin. Also included are CA15-3 antigen, M344antigen, Sialosyl Lewis Antigen (SLA), CA19-9, CA195 and other mucinantigen previously identified by monoclonal antibodies (e.g., see U.S.Pat. No. 5,849,876). The term mucin does not include proteoglycans whichare glycoproteins characterized by glycosaminoglycan chains covalentlyattached to the protein backbone.

At least 15 different mucins have been described including MUC1, MUC2,MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC12, MUC13,MUC15, and MUC16 (these may also be designated with a hyphen between“MUC” and the number). The nucleotide sequence and amino acid sequenceof these mucins are known. The NCBI and Swiss Prot accession nos. foreach of these mucins are as follows: MUC1 (NCBI NM002456, Swiss ProtP15941), MUC2, (NCBI NM002457, Swiss Prot Q02817) MUC3A (NCBI AF113616,Swiss Prot Q02505), MUC3B (NCBI AJ291390, Swiss Prot Q9H195), MUC4 (NCBINM138299, Swiss Prot Q99102), MUCSAC (NCBI AF043909, Swiss Prot Q8WWQ5),MUCSB (Swiss Prot Q9HC84), MUC6 (NCBI U97698, Swiss Prot Q8N8I1), MUC7(NCBI L42983, Swiss Prot Q8TAX7), MUC8 (NCBI U14383, Swiss Prot Q12964),MUC9 (NCBI U09550, Swiss Prot Q12889), MUC12 (Swiss Prot Q9UKN1), MUC13(NCBI NM017648, Swiss Prot Q9H3R2), MUC15 (NCBI NM145650, Swiss ProtQ8WW41), and MUC16 (NCBI AF361486, Swiss Prot Q8WXI7; aka CAl25).

There are two structurally and functionally distinct classes of mucins:secreted gel-forming mucins (MUC2, MUCSAC, MUCSB, and MUC6) andtransmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17). Theproducts of some MUC genes do not fit well into either class (MUC7,MUC8, MUC9, MUC13, MUC15, MUC16).

The characteristics of particular mucins as TAA in particular cancers issupported by alterations in expression and structure in association withpre-neoplastic and neoplastic lesions (Filipe MI: Invest Cell Pathol1979, 2:195-216; Filipe MI, Acta Med Port 1979, 1:351-365). Forinstance, normal mucosa of the stomach is characterized by theexpression of MUC1, MUCSA/C, MUC6 mRNA and the encoded immunoreactiveprotein. Also, high levels of MUC2, MUC3 mucin mRNA and encodedimmunoreactive protein are associated with intestinal metaplasia.Gastric cancer exhibits markedly altered secretory mucin mRNA levelscompared with adjacent normal mucosa, with decreased levels of MUCS andMUC6 mRNA and increased levels of MUC3 and MUC4 mRNA. High levels ofMUC2 and MUC3 mRNA and protein are detectable in the small intestine,and MUC2 is the most abundant colonic mucin.

Mucins represent diagnostic markers for early detection of pancreaticcancer and other cell types. Studies have shown, that ductaladenocarcinomas (DACs) and tumor cell lines commonly overexpress MUC1mucin . See Andrianifahanana et al., Clin Cancer Res 2001, 7:4033-4040).This mucin was detected only at low levels in the most chronicpancreatitis and normal pancreas tissues but is overexpressed in allstages of pancreatic cancers. The de novo expression of MUC4 inpancreatic adenocarcinoma and cell lines has been reported(Hollingsworth et al., Int J Cancer 1994, 57:198-203). MUC4 mRNAexpression has been observed in the majority of pancreaticadenocarcinoma and established pancreatic cancer cell lines but not innormal pancreas or chronic pancreatitis tissues. MUC 4 expression alsohas been associated with lung cancer (see Nguyen et al. 1996 Tumor Biol.17:176-192). MUCS is associated with metastases in non-small cell lungcancer (see Yu et al., 1996 Int. J. Cancer 69:457-465). MUC6 isoverexpressed and MUCSAC is de novo expressed in gastric and invasiveDACs (Kim et al., Gastroenterology 2002, 123:1052-1060). MUC7 has beenreported as a marker for invasive bladder cancer (see Retz et al. 1998Cancer Res. 58:5662-5666)

Expression of the MUC2 secreted gel-forming mucin is generally decreasedin colorectal adenocarcinoma, but preserved in mucinous carcinomas, adistinct subtype of colon cancer associated with microsatelliteinstability. MUC2 is increased in laryngeal cancer (Jeannon et al. 2001Otolaryngol Head Neck Surg. 124:199-202). Another secreted gel-formingmucin, MUCSAC, a product of normal gastric mucosa, is absent from normalcolon, but frequently present in colorectal adenomas and colon cancers.

MUC1, also known as episialin, polymorphic epithelial mucin (PEM), mucinlike cancer associated antigen (MCA), CA27.29, peanut-reactive urinarymucin (PUM), tumor-associated epithelial mucin, epithelial membraneantigen (EMA), human milk fat globule (HMFG) antigen, MUC1/REP,MUC1/SEC, MUC1/Y, CD227, is the most well known of the mucins. The geneencoding MUC1 maps to 1q21-q24. The MUC1 gene contains seven exons andproduces several different alternatively spliced variants. The tandemrepeat domain is highly O-glycosylated and alterations in glycosylationhave been shown in epithelial cancer cells.

MUC1 mRNA is polymorphic in size. There are presently nine isoforms ofMUC1 based on alternate splicing (isoform no.: NCBI accession no.; 1: IDP15941-1, 2: ID P15941-2, 3: ID P15941-3, 4: ID P15941-4, 5: P15941-5,6: ID P15941-6, 7: ID P15941-7, 8: ID P15941-8, and 9: ID P15941-9).

MUC1 isoform 1 (aka. MUC1/REP) is a polymorphic, type I transmembraneprotein containing: 1) a large extracellular domain, primarilyconsisting of a 20-amino acid (aa) repeat motif (a region known asVariable Number (30-100) of tandem repeats—VNTR); 2) a transmembranedomain; and 3) a 72-aa cytoplasmic tail. During biosynthesis, theMUC1/REP protein is modified to a large extent, and a considerablenumber of O-linked sugar moieties confer mucin-like characteristics onthe mature protein. Soon after translation, MUC1/REP is cleaved into twoproducts that form a tightly associated heterodimer complex composed ofa large extracellular domain, linked noncovalently to a much smallerprotein including the cytoplasmic and transmembrane domains. Theextracellular domain can be shed from the cell. Using Swiss Prot P15941as a reference (see FIG. 1), the extracellular domain (ecm) of MUC1isoform 1 represents amino acids 24 to 1158, the transmembrane domainrepresents 1159-1181, and the cytoplasmic domain represents 1182-1255.The SEA domain represents is 1034-1151 and represents a C-terminalportion of what is referred to as the extracellular domain. The SEAdomain of a mucin is generally a target for proteolytic cleavage,yielding two subunits, the smaller of which is associated with the cellmembrane.

MUC1 isoform 5 (aka MUC1/SEC) is a form of MUC1 that is secreted bycells. It has an extracellular domain that is identical to that ofisoform 1 (MUC1/REP), but lacks a transmembrane domain for anchoring theprotein to a cell membrane. MUC1 isoform 7 (aka MUC1/Y) contains thecytoplasmic and transmembrane domains observed in isoforms 1 (MUC1/REP)and 5 (MUC1/SEC), but has an extracellular domain that is smaller thanMUC1, lacking the repeat motif and its flanking region (see Baruch A. etal., 1999 Cancer Res. 59, 1552-1561). Isoform 7 behaves as a receptorand binds the secreted isoform 5. Binding induces phosphorylation ofisoform 7 and alters cellular morphology and initiates cell signalingthrough second messenger proteins such as GRB2, (see Zrihan-Licht S. etal., 1995 FEBS Lett. 356, 130-136). It has been shown that B-catenininteracts with the cytoplasmic domain of MUC1 (Yamamoto M. et al., 1997J. Biol. Chem. 272, 12492-12494).

MUC1 is expressed focally at low levels on normal epithelial cellsurfaces. See 15. Greenlee, et al., Cancer Statistics CA Cancer J. 50,7-33 (2000); Ren, et al., J. Biol. Chem. 277, 17616-17622 (2002);Kontani, et al., Br. J. Cancer 84, 1258-1264 (2001); Rowse, et al.,Cancer Res. 58, 315 (1998). MUC1 is overexpressed in carcinomas of thebreast, ovary, pancreas as well as other carcinomas (see also Gendler S.J. et al, 1990 J. Biol. Chem. 265, 15286-15293). A correlation is foundbetween acquisition of additional copies of MUC1 gene and high mRNAlevels (p<0.0001), revealing the genetic mechanism responsible for MUC1gene overexpression, and supporting the role of MUC1 gene dosage in thepathogenesis of breast cancer (Bièche I. et al.,. 1997 Cancer Genet.Cytogenet. 98, 75-80). MUC1 mucin, as detected immunologically, isincreased in expression in colon cancers, which correlates with a worseprognosis and in ovarian cancers.

High level expression of the MUC1 antigen plays a role in neoplasticepithelial mucosal cell development by disrupting the regulation ofanchorage dependent growth (disrupting E-cadherin function), which leadsto metastases. See Greenlee, et al., Cancer Statistics CA Cancer J. 50,7-33 (2000); Ren, et al. J. Biol. Chem. 277, 17616-17622 (2002).Non-MHC-restricted cytotoxic T cell responses to MUC1 have been reportedin patients with breast cancer. See Kontani et al., Br. J. Cancer 84,1258-1264 (2001). Human MUC1 transgenic mice (“MUC-1.Tg”) have beenreported to be unresponsive to stimulation with human MUC1 antigen. SeeRowse, et al., Cancer Res. 58, 315 (1998). Human MUC1 transgenic miceare useful for evaluating the development of immunity to MUC1 as a selfantigen.

MUC1 protein and mRNA have been found in the ER-positive MCF-7 andBT-474 cells as well as in the ER-negative MDA-MB-231 and SK-BR-3 BCCcells. The mRNA Transcript level was higher in ER+ than in ER-celllines. MUC1 reacts with intracellular adhesion molecule-1 (ICAM-1). Atleast six tandem repeats of MUC1 are needed (Regimbald et al., 1996Cancer Res. 56,4244-4249). The tandem repeat peptide of MUC1 from T-47DBCC was found to be highly O-glycosylated with 4.8 glycosylated sitesper repeat, which compares to 2.6 sites per repeat for the mucin frommilk.

The term “mucin antigen” as used herein refers to the full length mucinor a portion of a mucin that contains an epitope characterized in beingable to elicit cellular immunity using a MUC-CD40L expression vectoradministered in vivo as described herein. A “mucin antigen” includes oneor more epitopes from the extracellular domain of a mucin such as one ormore of the tandem repeat motifs associated with the VNTR, or the SEAregion. A mucin antigen may contain the entire extracellular domain.Also included within the meaning of “mucin antigen” are variations inthe sequence including conservative amino acid changes and the likewhich do not alter the ability of the antigen to elicit an immuneresponse that crossreacts with a native mucin sequence.

The VNTR consists of variable numbers of a tandemly repeated peptidesequences which differ in length (and composition) according to agenetic polymorphism and the nature of the mucin. The VNTR may alsoinclude 5′ and 3′ regions which contain degenerate tandem repeats. Forexample, in MUC1, the number of repeats varies from 21 to 125 in thenorthern European population. In the U.S. the most infrequent allelescontains 41 and 85 repeats, while more common alleles have 60-84repeats. The MUC1 repeat has the general repeating peptide sequencePDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 3). Underlying the MUC1 tandem repeatis a genetic sequence polymorphism at three positions shown bolded andunderlined (positions 2, 3 and 13). The concerted replacement DT->ES(sequence variation 1) and the single replacements PQ (sequencevariation 2), P→A (sequence variation 3), and P→T (sequence variation 4)have been identified and vary with position in the domain (see Engelmannet al., 2001 J. Biol. Chem. 276:27764-27769). The most frequentreplacement DT ES occurs in up to 50% of the repeats. Table 1 shows someexemplary tandem repeat sequences.

TABLE 1  Mucin Tandem Repeat Sequences Mucin Tandem Repeat (SEQ ID NO:)Mucin source MUC1 PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 3) MammaryPDNKPAPGSTAPPAHGVTSA (SEQ ID NO: 51) Pancreatic MUC2PTTTPPITTTTTVTPTPTPTGTQT (SEQ ID NO: 4) Intestinal Tracheobronchial MUC3HSTPSFTSSITTTETTS (SEQ ID NO: 5) Intestinal Gall Bladder MUC4TSSASTGHATPLPVTD (SEQ ID NO: 6) Colon Tracheobronchial MUC5ACTTSTTSAP (SEQ ID NO: 7) Gastric Tracheobronchial MUC5BSSTPGTAHTLTMLTTTATTPTATGSTATP Tracheobronchial (SEQ ID NO: 8) SalivaryMUC7 TTAAPPTPSATTPAPPSSSAPG (SEQ ID NO: 9) Salivary MUC8TSCPRPLQEGTPGSRAAHALSRRGHRVHELPTS TracheobronchialSPGGDTGF (SEQ ID NO: 10)

Although a mucin antigen as used herein may comprise only a singletandem repeat sequence motif, it should be understood that the immuneresponse will generally be stronger and more efficiently generated ifthe vector encodes multiple such repeats. The invention vectorpreferably encodes mucin tandem repeats from 2-4, more preferably from5-9, even more preferably from 10-19, yet even more preferably from20-29, still more preferably from 30-39, and still yet more preferablyfrom 40-50. Tandem repeats greater than 50 are possible and may includethe number of such repeats found in natural mucins.

A mucin antigen as this term is used herein also may encompass tandemrepeats from different types of mucins. For example, an expressionvector may encode tandem repeats from two different mucins, e.g., MUC1and MUC2. Such a vector also may encode multiple forms of the SEA domainas well or a combination of tandem repeats and one or more SEA domains.

A secretable form of an antigen is one that lacks all or substantiallyall of its transmembrane domain, if present in the mature protein. Forexample, in the case of a mucin, the transmembrane domain, if present,is generally about 24 amino acids in length and functions to anchor themucin or a fragment of the mucin in the cell membrane. A secretable formof MUC1 in which all of the transmembrane domain has been deleted isMUC1 missing residues 1159-1181. A mucin (or antigen) missingsubstantially all of the transmembrane is one where the domain comprises6 residues or less of sequence at one end of the transmembrane domain,more preferably less than about 4 residues of sequence at one end of thetransmembrane domain, even more preferably less than about 2 residues ofsequence on one end of the transmembrane domain, and most preferably 1residue or less on one end of the transmembrane domain In a preferredembodiment, the vaccine vector transcription unit encodes a secretableform of a mucin (or antigen) lacking the entire transmembrane domain Amucin that lacks substantially all of the transmembrane domain renderingthe mucin secretable is one that contains no more than six residues ofsequence on one end of the domain. The extracellular domain of a humanmucin such as MUC1 is denoted herein as “ecdhMUC 1.”

It should be understood that a mucin which lacks a functionaltransmembrane domain may still include all or a portion of thecytoplasmic domain and all or a portion of the SEA region, if present.

A source of DNA encoding the various mucins, and mucin antigens may beobtained from mucin expressing cell lines using a commercial cDNAsynthesis kit and amplification using a suitable pair of PCR primersthat can be designed from the published mucin DNA sequences. Forexample, MUC1 or MUC2 encoding nucleic acid may be obtained fromCRL-1500 cells, available from the American Type Culture Collection.Mucin encoding DNA also may be obtained by amplification from RNA orcDNA obtained or prepared from human or other animal tissues. For DNAsegments that are not that large, the DNA may be synthesized using anautomated oligonucleotide synthesizer.

The term “linker” as used herein with respect to the transcription unitof the expression vector refers to one or more amino acid residuesbetween the carboxy terminal end of the antigen and the amino terminalend of CD40 ligand. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. See e.g. Arai et al., design of the linkers whicheffectively separate domains of a bifunctional fusion protein. ProteinEngineering, Vol. 14, No. 8, 529-532, August 2001. The linker isgenerally from about 3 to about 15 amino acids long, more preferablyabout 5 to about 10 amino acids long, however, longer or shorter linkersmay be used or the linker may be dispensed with entirely. Longer linkersmay be up to about 50 amino acids, or up to about 100 amino acids. Ashort linker of less than 10 residues is preferred when the mucinantigen is N-terminal to the CD40 ligand.

The term “CD40 ligand” (CD40L) as used herein refers to a full length orportion of the molecule known also as CD 154 or TNFS. CD40L is a type IImembrane polypeptide having a cytoplasmic domain at its N-terminus, atransmembrane region and then an extracellular domain at its C-terminus.Unless otherwise indicated the full length CD40L is designated herein as“CD40L,” “wtCD40L” or “wtTmCD40L.” The form of CD40L in which thecytoplasmic domain has been deleted is designated herein as “ΔCtCD40L.”The form of CD40L where the transmembrane domain has been deleted isdesignated herein as “ΔTmCD40L.” The form of CD40L where both thecytoplasmic and transmembrane domains have been deleted is designatedherein as “ΔCtΔTmCD40L.” The nucleotide and amino acid sequence of CD40Lfrom mouse and human is well known in the art and can be found, forexample, in U.S. Pat. No. 5,962,406 (Armitage et al.). Also includedwithin the meaning of CD40 ligand are variations in the sequenceincluding conservative amino acid changes and the like which do notalter the ability of the ligand to elicit an immune response to a mucinin conjunction the fusion protein of the invention.

Murine CD40L (mCD40L) is 260 amino acids in length. The cytoplasmic (Ct)domain of mCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-260.

Human CD40L (hCD40L) is 261 amino acids in length. The cytoplasmicdomain of hCD40L extends approximately from position 1-22, thetransmembrane domain extends approximately from position 23-46, whilethe extracellular domain extends approximately from position 47-261.

The phrase “CD40 ligand is missing all or substantially all of thetransmembrane domain rendering CD40 ligand secretable” as used hereinrefers to a recombinant form of CD40 ligand that can be secreted from acell. The transmembrane domain of CD40L which contains about 24 aminoacids in length, functions to anchor CD40 ligand in the cell membrane.CD40L from which all of the transmembrane domain has been deleted isCD40 ligand lacking residues 23-46. CD40 ligand missing substantiallyall of the transmembrane is one that retains 6 residues or less ofsequence at one end of the transmembrane domain, more preferably lessthan about 4 residues of sequence at one end of the transmembranedomain, even more preferably less than about 2 residues of sequence onone end of the transmembrane domain, and most preferably 1 residue orless on one end of the transmembrane domain Thus, a CD40L that lackssubstantially all of the transmembrane domain rendering the CD40Lsecretable is one that retains no more than six residues of sequence onone end of the domain. Such as CD40L would contain, in addition to theextracellular domain and optionally the cytoplasmic domain, and no morethan amino acids 41-46 or 23-28 located in the transmembrane domain ofCD40L. In a preferred embodiment, the vaccine vector transcription unitencodes a secretable form of CD40 containing less than 10% of thetransmembrane domain. More preferably, CD40L contains no transmembranedomain

It should be understood that a CD40L which lacks a functionaltransmembrane domain may still include all or a portion of thecytoplasmic domain. Likewise, a CD40L which lacks a functionaltransmembrane domain may include all or a substantial portion of theextracellular domain.

As used herein, an expression vector and fusion protein boost isadministered as a vaccine to induce immunity to a tumor antigen. Theexpression vector and protein boost may be formulated as appropriatewith a suitable pharmaceutically acceptable carrier. Accordingly, thevectors or protein boost may be used in the manufacture of a medicamentor pharmaceutical composition. Expression vectors and the fusion proteinmay be formulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use.Liquid formulations may be buffered, isotonic, aqueous solutions.Powders also may be sprayed in dry form. Examples of suitable diluentsare normal isotonic saline solution, standard 5% dextrose in water, orbuffered sodium or ammonium acetate solution. Such formulations areespecially suitable for parenteral administration, but may also be usedfor oral administration or contained in a metered dose inhaler ornebulizer for insufflation. It may be desirable to add excipients suchas polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,polyethylene glycol, mannitol, sodium chloride, sodium citrate, and thelike.

Alternately, expression vectors and the fusion protein may be preparedfor oral administration. Pharmaceutically acceptable solid or liquidcarriers may be added to enhance or stabilize the composition, or tofacilitate preparation of the vectors. Solid carriers include starch,lactose, calcium sulfate dihydrate, terra alba, magnesium stearate orstearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriersinclude syrup, peanut oil, olive oil, saline and water. The carrier mayalso include a sustained release material such as glyceryl monostearateor glyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably, will be between about 20 mg to about 1 g perdosage unit. When a liquid carrier is used, the preparation may be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension.

Expression vectors and the fusion protein may be formulated to includeother medically useful drugs or biological agents. The vectors also maybe administered in conjunction with the administration of other drugs orbiological agents useful for the disease or condition that the inventioncompounds are directed.

As employed herein, the phrase “an effective amount,” refers to a dosesufficient to provide concentrations high enough to generate (orcontribute to the generation of) an immune response in the recipientthereof. The specific effective dose level for any particular subjectwill depend upon a variety of factors including the disorder beingtreated, the severity of the disorder, the activity of the specificcompound, the route of administration, the rate of clearance of theviral vectors, the duration of treatment, the drugs used in combinationor coincident with the viral vectors, the age, body weight, sex, diet,and general health of the subject, and like factors well known in themedical arts and sciences. Various general considerations taken intoaccount in determining the “therapeutically effective amount” are knownto those of skill in the art and are described, e.g., in Gilman et al.,eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Co., Easton, Pa., 1990. For administration ofvectors, the range of particles per administration typically if fromabout 1×10⁷ to 1×10¹¹ , more preferably 1×10⁸ to 5×10¹⁰, and even morepreferably 5×10⁸ to 2×10¹⁰. A vector can be administered parenterally,such as intravascularly, intravenously, intraarterially,intramuscularly, subcutaneously, or the like. Administration can also beorally, nasally, rectally, transdermally or inhalationally via anaerosol. The vectors may be administered as a bolus, or slowly infused.The vector is preferably administered subcutaneously.

As demonstrated herein, vectors encoding tumor associated antigens caninduce a protective cellular and humoral immunity against such antigens,including those to which tolerance had developed. Although not wishingto be bound by any theory, it is believed that the invention vaccinesgenerate upon administration a continual local release of the fusionprotein composed of the secretable form of the antigen linked to asecretory form of CD40 ligand. As demonstrated herein this facilitatesDCs maturation, promoting the development of effective antigen-specificimmunity. It is also demonstrated herein that the secretable fusionprotein encoding the extracellular domain of human MUC1 and the murineCD40L lacking a transmembrane and cytoplasmic domain (i.e.ecdhMUC1-ΔCtΔTmCD40L) produced from an adenoviral vector dramaticallyenhanced the potency of the cellular immune response to MUC1 expressingtumor cells. Although not wishing to be bound by any theory, it isbelieved that subcutaneous injection of the Ad-K-ecdhMUC1-ΔCtΔTmCD40Lvector elicited strong MUC1 specific CD8⁺ T cell-mediated immunity,which prevents the engraftment of cancer cells which express the MUC1tumor associated antigen.

The immunity generated against the antigens using the invention methodsis long lasting. As used herein, the term long lasting means thatimmunity elicited by the antigen encoded by the vector can bedemonstrated for up to 6 months from the last administration, morepreferably for up to 8 months, more preferably for up to one year, morepreferably up to 1.5 years, and more preferably for at least two years.

In one embodiment, immunity to a mucin TAA can be generated by producinga fusion protein that comprises the extracellular domain of MUC1 fusedthe amino-terminal end of the CD40 ligand from which the transmembraneand cytoplasmic domains were deleted. Construction of such vector isdisclosed in the Examples. As was observed herein, subcutaneousadministration of this adenoviral vector mucin vaccine induced a veryrobust and long lasting CD8⁺ cytotoxic T cell lymphocyte dependentsystemic immune response against cancer cells which carry the MUC1antigen. The mucin vaccine induced the production of memory cells, whichunderlie the long lasting immunity.

It was observed that vaccination of mice with the adenoviral vectorAd-sig-ecdhMUC1/ecdmCD40L induced an immune response which suppressedthe growth of human MUC1 (hMUC1) antigen positive tumor cells in 100% ofmice transgenic for hMUC1 (i.e. these mice are anergic to the hMUC1antigen prior to the vector injection. See Rowse, et al., Cancer Res.58, 315 (1998). The immune response to the Ad-sig-ecdhMUC1/ecdmCD40Lvector lasted up to a year and was shown to be antigen specific. Theseresults demonstrated that the Ad-sig-ecdhMUC1-ecd/ecdCD40L vector can beused for treating epithelial malignancies that express the MUC1.

Subcutaneous injection of the adenoviral MUC1 expression vectorincreased the level of hMUC1 specific T cells in the spleens of injectedhMUC1 transgenic mice by 250 fold. The transgenic mice were allergic tothe hMUC1 antigen prior to the vector injection. Thus, vector injectionovercame the anergy, inducing a CD8+ T cell dependent systemic Thlimmune response that was antigen specific, and HLA restricted. Theability to overcome anergy as observed for vaccination with theadenoviral MUC1 expression vector, was not observed when transgenic micewere vaccinated with purified ecdhMUC1/ecdCD40L-HIS protein.

Although not wishing to be bound by any theory, it is believed that thecells infected in the vicinity of the site of subcutaneous injection ofthe vector release the tumor antigen/CD40 ligand secretory which istaken up by antigen presenting cells (e.g. DCs) in the vicinity of theinfected cells. The internalized tumor antigen would be digested in theproteosome with the resultant tumor antigen peptides trafficking to theendoplasmic reticulum where they would bind to Class I MHC molecules.Eventually, the DCs would present the tumor antigen on the surface inthe Class I MHC molecule. Activated, tumor antigen-loaded antigenpresenting cells would migrate to lymphocyte bearing secondary organssuch as the regional lymph nodes or the spleen. During the two weeks ofcontinuous release of the tumor antigen/CD40 fusion protein, CD8cytotoxic T cell lymphocytes competent to recognize and kill cells,which carried the tumor associated antigens, would be expanded in thelymph nodes and spleen by the presence of the activated and antigenloaded dendritic cells. The continuous nature of the stimulation and theexpansion of the tumor antigen specific cytotoxic T cells by thecontinuous release from the vector infected cells is believed togenerate an immune response which would be greater in magnitude than ispossible using a vector which carried a tumor antigen/CD40 ligand whichis non-secretory.

The methods of the present invention, therefore, can be used to generateimmunity to an antigen which is a self-antigen in an individual. Forexample, a vector that encodes a mucin antigen from MUC1 can be used togenerate CD8⁺ immunity in a human where the MUC1 mucin antigen is a selfantigen. The invention methods also can be used to overcome a state ofimmunological anergy to an antigen which is a self-antigen.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES

1. Construction of Adenoviral Expression Vectors

The transcription unit, sig-ecdhMUC1-ΔCtΔTmCD40L of the adenoviralvector encodes a signal sequence (from an Ig kappa chain) followed bythe extracellular domain of human MUC1 which is connected via a linkerto a fragment of the CD40 ligand (human or mouse) which contains theextracellular domain without the transmembrane or cytoplasmic domains.The fusion protein was engineered to be secreted from vector infectedcells by the addition of the kappa chain signal sequence to theamino-terminal end of the fusion protein.

The amino acid sequence of human MUC-1 and the encoding nucleotidesequence are shown in FIGS. 2 and 1, respectively. The encoded MUC1protein represents 1255 amino acids encoded by nucleotides 74 to 3,841of SEQ ID NO: 1. The first 23 amino acids (encoded by 74 to 142 of SEQID NO:1) represent the MUC1 signal sequence which is removed from themature mucin. The extracellular domain represents about 1135 amino acidsfrom positions 24 to 1158 (encoded by nucleotides 143 to 3547). Thetandem repeat region represents approximately 900 amino acids. Aminoacids 74 to 126 (encoded by 229 to 451 of SEQ ID NO:1) represents a 5′degenerate tandem repeat region, amino acids 127 to 945 represents thetandem repeat region (encoded by 452 to 2,908 of SEQ ID NO: 1) whileamino acids 946 to 962 represent a 3′ degenerate tandem repeat region(encoded by 2809 to 2959 of SEQ ID NO:1). The SEA domain representsamino acids 1034 to 1151, the transmembrane domain represents 1159 to1181, and the cytoplasmic domain represents 1182 to 1255 (see SEQ IDNO:2).

The transcription unit was introduced into the E1 gene region of theadenoviral vector backbone. After the adenoviral vector particles weregenerated in HEK 293 cells, the vector DNA was purified by cesiumchloride gradient centrifugation. The presence of the signal peptide inthe adenoviral vector was confirmed by restriction enzyme analysis andby DNA sequencing.

A transcription unit that included DNA encoding the signal sequence ofthe mouse IgG kappa chain gene upstream of DNA encoding human MUC-1(“sig-ecdhMUC-1”) was generated by PCR using plasmid pcDNA3-hMUC-1 (giftof Finn O.J., University of Pittsburgh School of Medicine) and thefollowing primers: DNA encoding the mouse IgG kappa chainMETDTLLLWVLLLWVPGSTGD (single letter amino acid code) (SEQ ID NO: 11)was prepared by PCR amplification (SEQ ID NOs: 12 ,13 and 14) togenerate the full 21 amino acid mouse IgG kappa chain signal sequence(the start codon “ATG” is shown bolded in SEQ ID NO:12).

(SEQ ID NO: 12) 5′-CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTACTG CTG-3′ (SEQ ID NO: 13)5′-TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3′ (SEQ ID NO: 14)5′-TG CTC TGG GTT CCA GGT TCC ACT GGT GAC GAT G-3′ (SEQ ID NO: 15) 5′-GGT TCC ACT GGT GAC GAT GTC ACC TCG GTC CCA GTC-3′(forward primer for MUC-1 repeat region) (SEQ ID NO: 16) 5′-GAGCTCGAG ATT GTG GAC TGG AGG GGC GGT G-3′(reverse primer for MUC-1 repeat region)sig-ecdhMUC-1 with the upstream kappa signal sequence was generated byfour rounds of PCR amplification (1″ round: primers SEQ ID NOs 15 and16; 2^(nd) round: primer SEQ ID NOs 14 and 16; 3r^(d) round: primer SEQID NOs 13 and 16; 4^(th) round: primer SEQ ID NOs 12 and 16). Thesig-ecdhMUC-1 encoding DNA was cloned into the pcDNA™ 3.1 TOPO vector(Invitrogen, San Diego, Calif.) forming pcDNA-sig-ecdhMUC-1.

pShuttle -ΔCtΔTmCD40L (no signal sequence and murine CD40L) was preparedas follows: Plasmid pDC406-mCD40L was purchased from the American TypeCulture Collection. A pair of PCR primers (SEQ ID NOs: 17 and 18) wasdesigned to amplify the mouse CD40 ligand from position 52 to 260 (i.e.,without the cytoplasmic and transmembrane domains) and include sequenceencoding a linker (indicated as “+spacer”) at the 5′ end of theamplicon.

Mouse ΔCtΔTmCD40L + spacer forward primer(MCD40LSPF) (CD40L sequence italicized; cloningsite underlined and bolded): (SEQ ID NO: 17)5′-CCGCTCGAGAACGACGCACAAGCACCAAAATCAAAGGTCGAAG AGGAAGTA-3′.Mouse CD40L reverse primer (MCD40LR; cloning site underlined)(SEQ ID NO: 18) 5′-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAG TAAGCC AAA AGA TGA G-3′

The forward primer MCD40LSPF encodes a 10 residue spacer (LENDAQAPKS;single letter code; SEQ ID NO: 19) to be located between the mucin andthe CD40 ligand (mCD40L) of the transcription unit. PCR performed usingthe forward and reverse primers (SEQ ID NOs 17 and 18) and plasmidpDC406-mCD40L as the template resulted in PCR fragment“space+ΔCtΔTMCD40L”, which was inserted into the plasmidpcDNA-sig-ecdhMUC1 after restriction endonuclease digestion with XbaI(TCTAGA) and Xho I (CTCGAG). This vector is designatedpcDNA-sig-ecdhMUC1/ΔCtΔTmCD40L. A vector was produced that was otherwisethe same except that it encoded full length CD40L rather than thetruncated form. This vector was made using a CD40 forward primer thatannealed to the starting codons of murine CD40L. This vector isdesignated pShuttleCD40L (no signal sequence).

The sig-ecdhMUC1/ΔCtΔTmCD40L encoding DNA was cut from the pCDNA3TOPOvector using HindIII-XbaI restriction and inserted into pShuttle-CMV(see Murphy et al., Prostate 38: 73-78, 1999) downstream of the CMVpromoter. The plasmid is designated pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40L.Thus, the transcription unit sig-ecdhMUC1-ΔCtΔTmCD40L encodes the mouseIgG kappa chain secretory signal followed by the extracellular domain ofhuman MUC1 followed by a 10 amino acid linker with (NDAQAPK; residues3-9 of SEQ ID NO: 19) followed by murine CD40 ligand residues 52-260.

In some vectors, the mouse HSF1 trimer domain was added between theecdhMUC1 encoding DNA and ΔCtΔTm CD40L by PCR using plasmidpcDNA-sig-ecdhMUC1/ΔCtΔTmCD40L and the following primers:

(SEQ ID NO: 20) 5′-AAC AAG CTC ATT CAG TTC CTG ATC TCA CTG GTGGGATCC AAC GAC GCA CAA GCA CCA AAA TC-3′. (SEQ ID NO: 21)5′-AGC CTT CGG CAG AAG CAT GCC CAG CAA CAG AAAGTC GTC AAC AAG CTC ATT CAG TTC CTG-3′. (SEQ ID NO: 22)5′AAT GAG GCT CTG TGG CGG GAG GTG GCC AGC CTT CGG CAG AAG CAT G-3′.(SEQ ID NO: 23) 5′GAT ATC CTC AGG CTC GAG AAC GAC GCA CAA GCACCA AAA GAG AAT GAG GCT CTG TGG CGG G-3′. (SEQ ID NO: 18)5′-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAG TAA GCC AAA AGA TGA G-3′.

HSF1/ΔCtΔTm CD40L with the trimer domain sequence was generated by fourrounds of PCR amplification (1^(st) round: primers SEQ ID NOs 23 and 18;2^(nd) round: primer SEQ ID NOs 22 and 18; 3^(rd) round: primer SEQ IDNOs 21 and 18; 4^(th) round: primer SEQ ID NOs 20 and 18). TheHSF1/ΔCtΔTm CD40L encoding DNA was cloned into pcDNA-sig-hMUC-1restriction sites XbaI (TCTAGA) and Xho I (CTCGAG). The sequence betweenMUC1 and mCD40L is as follows:

(SEQ ID NO: 24) L E N D A Q A P K E N E A L W R E V A S F R Q KH A Q Q Q K V V N K L I Q F L I S L V G S N D A Q A P K S,wherein the underlined segment is the trimer sequence which is bonded bythe linker LENDAQAPK (SEQ ID NO:25) and NDAQAPKS (SEQ ID NO:26) .

In some vectors, a His tag encoding sequence was added to the end of theΔCtΔTm CD40L and was generated by PCR using Plasmid pDC406-mCD40L(purchased from the American Type Culture Collection) and the followingprimers:

(forward primer) (SEQ ID NO: 27) 5′-CCG CTCGAGAACGACGCACAAGCACCAAAATCAAAGGTCGAAGAGG AAGTA-3′(reverse primer) (SEQ ID NO: 28) 5′-ATG GTG ATG ATG ACC GGT ACG GAG TTT GAG TAA GCCAAA AGA TGA GAA GCC-3′(poly His region encoded by nucleotides in the box) (SEQ ID NO: 29) 

Vector /ΔCtΔTm CD40L/His with the His tag sequence was generated by 2rounds of PCR amplification (1^(st) round: primers 1 +2; 2^(nd) round:primer 1+3). The /ΔCtΔTmCD40L/His encoding DNA was cloned intopcDNA-sig-ecdhMUC-1 restriction sites XbaI (TCTAGA) and Xho I (CTCGAG).

The recombinant adenoviral vectors were generated using the AdEasyvector system (Stratagene, San Diego, Calif.). Briefly the resultingplasmid pShuttle-sig-ecdhMUC1-ΔCtΔTmCD40L, and other control adenoviralvectors were linearized with Pme I and co-transformed into E. colistrain BJ5183 together with pAdEasy-1, the viral DNA plasmid.Recombinants were selected with kanamycin and screened by restrictionenzyme analysis. The recombinant adenoviral construct was then cleavedwith Pac Ito expose its Inverted Terminal Repeats (ITR) and transfectedinto 293A cells to produce viral particles. The titer of recombinantadenovirus was determined by the Tissue culture Infectious Dose (TCID₅₀)method.

Primers for amplifying human ΔCtΔTmCD40L+ spacer using a human CD40ligand cDNA template are set forth below.

Human ΔCtΔTmCD40L + spacer forward primer(HCD40LSPF) (CD40L sequence italicized): (SEQ ID NO: 30) 5′-CCGCTCGAGAACGACGCACAAGCACCAAAATCAGTGTATCTTCATAGAAGGTTG GACAAG-3′Human CD40L reverse primer (HCD40LR) (SEQ ID NO: 31)5′-CCCTCTAGA TCAGAGTTTGAGTAAGCCAAAGGAC-3′

These primers will amplify a ΔCtΔTmCD40L+spacer which encodes 47-261 ofhuman CD40L. The forward primer HCD40LSPF encodes a 10 residue spacer(LENDAQAPKS; single letter code; SEQ ID NO: 19) to be located betweenthe tumor antigen and the CD40 ligand (hCD40L) of the transcriptionunit. PCR performed using the forward and reverse primers (SEQ ID NOs 30and 31) and Plasmid pDC406-hCD40L as the template results in PCRfragment “space+ΔCtΔTmCD40L(human),” which is inserted into the plasmidpcDNA-sig-ecdhMUC1 after restriction endonuclease digestion with XbaI(TCTAGA) and Xho I (CTCGAG). The sig-ecdhMUC1/ΔCtΔTmCD40L (human)encoding DNA was cut from the pCDNA3TOPO using HindIII-XbaI restrictionand inserted into pShuttle-CMV (see Murphy et al., Prostate 38: 73-78,1999) downstream of the CMV promoter. This vector is designated pShuttlesig-ecdhMUC1/ΔCtΔTmCD40L(human). Modification of pShuttlesig-ecdhMUC1/ΔCtΔTmCD40L(human) to include the ecdhMUC1 upstream of thehuman CD40 ligand sequence was accomplished essentially as describedabove for the murine CD40 ligand encoding vectors. Thus, thetranscription unit sig-ecdhMUC1-ΔCtΔTmCD40L(human) encodes the kappasecretory signal followed by the extracellular domain of human MUC1followed by a 10 amino acid linker (NDAQAPK; residues 3-9 of SEQ IDNO:19) followed by human CD40 ligand residues 47-261.

In an alternative approach, DNA encoding the human growth hormone signalsequence MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQID NO: 32) could be used in place of the kappa chain signal sequence.

2. Overcoming Anergy to MUC1 in MUC1 Transgenic Mice

a) Cytokine Production of Adenoviral Infected DCs

Bone marrow derived DCs was harvested from hMUC-.Tg transgenic mice at48 hours after exposure to the adenoviral vectors. The cells wereexposed to vector at MOI 100, and plated in 24-well plates at 2×10⁵cells/ml. After incubation for 24 hours at 37° C., supernatant fluid (1ml) was harvested and centrifuged to remove debris. The level of murineIL-12 or IFN-gamma released into the culture medium was assessed byenzyme-linked immunoadsorbent assay (ELISA) using the mouse IL-12 p70 orIFN-gamma R & D Systems kits.

Bone marrow derived DCs contacted with the Ad-sig-ecdmMUC1-ΔCtΔTCD40L(murine) vector showed significantly increased the levels of interferongamma and IL-12 cytokines from DCs harvested from the hMUC-.Tgtransgenic mice at 48 hours after exposure to the vector. In contrast,virtually no cytokines were detected from restimulated DC's from animalsimmunized with an adenoviral vector that encoded the extracellulardomain of hMUC 1 but without fusion to a secretable form of CD40L. Theseresults indicate that the ecdhMUC1/ecdmCD40L (murine) fusion proteinforms functional trimers and binds to the CD40 receptor on DCs.

b) Evaluation of Trimer Formation by ecdhMUC1-HSF1-ΔCtΔTmCD40L FusionProtein Expressed from Ad-sig-ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS

Trimerization of ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS fusion protein wasevaluated following release from cells transformed withAd-sig-ecdhMUC1-HSF1-ΔCtΔTmCD40L-HIS vector. The expressed fusionprotein was purified from the supernatant of 293 cells exposed to thevector using a His Tag purification kit. Nondenaturing gelelectrophoresis showed a molecular weight consistent with trimerformation.

c) Effect of Ad-sig-ecdhMUC1-ΔCtΔTmCD40L Vector Injection onEstablishment of MUC1 Expressing Cancer Cells.

hMUC-1.Tg mice injected subcutaneously with theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector were resistant toengraftment by the hMUC1 positive LL2/LL1hMUC1 mouse cancer cells.Control animals not injected with vector were not resistant to thegrowth of the same cells. Also, hMUC-1.Tg mice injected with theAd-sig-ecdhMUC1/ecdCD40L (murine) vector were not resistant toengraftment by parental cell line (LL2/LL1), which does not expressMUC1.

hMUC-1.Tg mice injected intravenously with ecdhMUC1-ΔCtΔTmCD40L (murine)protein were not resistant to engraftment by the hMUC1 positiveLL2/LL1hMUC1 mouse cancer cells. Furthermore, hMUC-1.Tg mice injectedwith Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector lived longer than didcontrol vector injected mice subsequently administered the LL2/LL1hMUC1cell line.

3. Cellular Mechanisms Underlying Breakdown of Anergy

a) Cytokine Release from Vaccinated vs. Non Vaccinated Mice.

A population of splenic CD8⁺ T lymphocytes was obtained seven daysfollowing Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector administration wasobtained by depleting CD4⁺ T lymphocytes using CD4⁺ antibody coatedmagnetic beads. The isolated CD8⁺ T lymphocytes released over 2,000times the level of interferon gamma as did CD8⁺ T cells from MUC-1.Tgmice administered a control vector (without MUC1).

b) Cytotoxicity Assay

Splenic T cells collected from hMUC-1.Tg mice 7 days followingadministration of Ad-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector werecultured with hMUC1 antigen positive LL2/LL1hMUC1 cancer cells in vitrofor 7 days. The stimulated splenic T cells were mixed in varying ratioswith either the hMUC1 positive LL2/LL1hMUC1 cells or the hMUC1 negativeLL2/LL1 cancer cells. The results showed that T cells fromAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector vaccinated mice werecytotoxic only for the cancer cells expressing hMUC1.

c) Ad-sig-ecdhMUC1-ΔCtΔTmCD40L vector Injection Overcomes Resistance toExpansion of hMUC1 Specific T Cells.

DCs obtained in vitro from bone marrow cells were exposed to theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector for 48 hours. Splenic CD8⁺ Tcells, obtained from hMUC-1.Tg transgenic mice 7 days following novector injection or subcutaneous injection with theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector, were mixed in a 1/1 ratiowith the Ad-sig-ecdhMUC1/ecdCD40L (murine) vector-infected DCs. TheERK1/EK2 proteins, the endpoint of the Ras/MAPK signaling pathway, werephosphorylated in the CD8+ T cells isolated fromAd-sig-ecdhMUC1-ΔCtΔTmCD40L vector injected hMUC-1.Tg transgenic micefollowing 45 minutes of in vitro exposure to Ad-sig-ecdhMUC1-ΔCtΔTmCD40L(murine) vector infected DCs. In contrast no increase in phosphorylationof ERK1 and ERK2 proteins was seen in CD8 positive T cells fromunvaccinated hMUC-1.Tg mice. These results demonstrate that CD8 positiveT cells from MUC-1.Tg transgenic mice vaccinated with theAd-sig-ecdhMUC1-ΔCtΔTmCD40L (murine) vector were no longer anergic toMUC1.

4. Production of the Fusion Protein and Vector

The tumor antigen fusion protein was produced directly from anadenoviral vector that carries the expression cassette of the fusiongene encoding the fusion protein. The production cells (e.g. 293 cellline) at 80% confluency in growth medium were infected with the viralvector at the ratio of 10-100 viral particles per cell. The infectedcells were further cultured for 48-72 hours, when the viral vectorspropagated in the cells and the tumor antigen fusion proteins wereexpressed in the cells and secreted into culture media. The infectedcells were collected when 70-90% of them showed cytopathic effect (CPE).The cell culture media was collected separately. Cell lysates wereprepared through 3-time freeze-and-thaw cycles. The viral particles wereisolated via the standard procedure (19). The tumor antigen fusionproteins were purified through affinity chromatograph from the collectedcell media 5. Amplification of the Immune Response by Protein Boosting

The relative value of protein boosting with the tumor antigen fusionprotein versus boosting with the adenoviral expression vector wasevaluated.

hMUC-1.Tg animals were primed by subcutaneous administration ofAd-K/ecdhMUC1-ΔCtΔTmCD40L vector as described. The protein boostconstituted 10 micrograms of ecdhMUC-1/ecdCD40L fusion protein injectedsubcutaneously. The time of protein boosting and comparison with vectorwas evaluated in various treatment groups shown in table 2.

TABLE 2 Immunization Schedule Testing Group Week 1 Week 2 Week 3 Week 4Control Vector Vector Nothing Nothing Treatment 1 (T1) Vector VectorProtein Nothing Treatment 2 (T2) Vector Vector Nothing Protein Treatment3 (T3) Vector Protein Nothing Nothing Treatment 4 (T4) Vector NothingProtein Nothing Treatment 5 (T5) Vector Protein Nothing Protein NegativeControl Nothing Nothing Nothing Nothing

Spleen cells from the different groups were isolated and evaluated bythe ELISPOT assay for interferon gamma positivity. As seen in FIG. 3.two subcutaneous protein injections at a 14 day interval beginning oneweek after the initial vector injection showed the greatest elevation ofthe frequency of positive T cells as compared to no treatment orcompared with one or two vector injections without protein boost. Thenext highest elevation of the frequency of interferon gamma positive Tcells was with the T3 group (one protein injection 7 days following theinitial vector injection).

Cytotoxic T cells development in the various immunization groups wasalso evaluated (FIG. 4). Spleen cells from the various treatment groupswere stimulated in vitro for 5 days with a hMUC-1 positive cell line(LL1/LL2hMUC-1). CD8 T cells were isolated and mixed with the targetcells (LL1/LL2hMUC-1) in a 50/1 ratio. Cytotoxic activity generallyfollowed the ELISPOT assay results, with the T5 group showing thegreatest increase levels of LL1/LL2hMUC-1 specific cytotoxic T cellactivity. The level of cytotoxicity seen with T cells from the T5 groupwas nine fold that seen with the negative control group.

Serum from the animals in the various treatment groups were evaluatedfor anti-ecdhMUC1-ΔCtΔTmCD40L specific antibodies in an ELISA. Briefly,microwells coated with the ecdhMUC1-ΔCtΔTmCD40L protein were incubatedwith test mouse serum, washed and bound mouse antibody identified usinga secondary rat anti-mouse antibody conjugated to horseradishperoxidase.

FIG. 5 shows a dramatic increase in the level of antibodies to theecdhMUC1-ΔCtΔTmCD40L fusion protein generated by the treatment with onevector injection and two protein injections spaced at a 14 day interval.The increase in the anti-ecdhMUC1-ΔCtΔTmCD40L antibodies following theT5 treatment was 2 fold greater than with any of the other treatmentgroup.

The results from these assays demonstrate that protein boosting issuperior to vector boosting in generating cytotoxic T cell activityagainst tumor antigen expressing cells as well as antibody responses tothe tumor antigen. The overall best results with protein boosting wereobtained using a single injection of adenoviral expression vectorfollowed one week later with a subcutaneous protein boost, which isrepeated two weeks later by another protein boost.

Antibodies in serum from vaccinated hMUC-1.Tg mice were evaluated forbinding to cancer biopsy tissue specimens. Tissue microarrays containingnormal breast and breast cancer tissue sections were obtainedcommercially. Tissue was contacted with serum from transgenic miceimmunized with Ad-K/ecdhMUC-1/ΔCtΔTm CD40L vector and boosted later withecdhMUC-1/ΔCtΔTm CD40L protein. The arrays were washed and then exposedto a horseradish peroxidase (HRP) secondary antibody which recognizesmouse IgG antibody. As a control, the serum was exposed first to ahMUC-1 peptide from the antigenic repeat of the hMUC-1 domain (same asused for the protein boost).

Serum from the vaccinated mice bound to the breast epithelial cells frombiopsy specimens of cancerous epithelial cells. No binding to theintervening fibroblast or stromal cells were observed. Serum from normalmice showed no reaction.

Serum from hMUC-1.Tg mice vaccinated with the Ad-sig-hMUC-1/ecdCD40Lfollowed by two subsequent administrations of protein sc-hMUC-1/ecdCD40Lreacted with biopsy specimens from human prostate cancer on tissuemicroarray slides.

To determine specificity of the serum generated antibodies for thehMUC-1 repeat, serum from vaccine immunized animals described above wasmixed with increasing amounts of a peptide containing the amino acidsequence from the hMUC-1 repeat. The mixture was then applied to themicroarray slides and evaluated for reactivity. A peptide with the sameamino acids as the hMUC-1 repeat but with the sequence scrambled(“scrambled peptide”) was added to serum from vaccinated animals as acontrol. The hMUC-1 peptide blocked binding of the antibodies invaccinated serum to the breast cancer epithelial cells. No blocking wasseen for the scrambled peptide. These suggests demonstrate that thevector prime/protein boost vaccination induced a hMUC-1 specific humoralresponse reactive with MUC-1 expressed by biopsy specimens of humanbreast cancer epithelial cells.

Tumor immunity in protein boosted mice was evaluated. hMUC-1.Tg animalswere primed by subcutaneous administration of Ad-K/ecdhMUC1-ΔCtΔTmCD40Lvector as described or were immunized with one or two administrations ofthe ecdhMUC1-ΔCtΔTmCD40L fusion protein. Animals were then challengedwith LL2/LL1hMUC-1 tumor cells.

FIG. 6 shows that mice vaccinated with the Ad-K/ecdhMUC1-ΔCtΔTmCD40Lvector survived longer than 120 days (solid bold line), whereas all micenot vaccinated with the Ad-sig-ecdhMUC-1/ecdCD40L vector died by 50 days(broken line). These results show that the vector injections induced asuppression of the growth of the LL2/LL1hMUC-1 cell line in thehMUC-1.Tg mice.

The specificity of tumor growth suppression for the hMUC-1 antigen wasevaluated by comparing rejection of the LL2/LL1hMUC-1 cell line (whichis positive for the hMUC-1 antigen) with the LL2/LL1 cell line, which isotherwise identical except for the absence of the hMUC-1 antigen. Theresults showed subcutaneous injection of the adenoviral vectorcompletely suppressed the growth of the LL2/LL1hMUC-1 cell line but didnot the same cells which do not express MUC-1.

Tumor growth suppression was evaluated using combinations of vector andprotein administration. Three combinations of Ad-sig-ecdhMUC-1/ecdCD40Lvector and ecdhMUC-1/ecdCD40L protein were administered to hMUC-1.Tgmice before challenge with LL2/LL1hMUC-1 tumor cells. VVV=threeAd-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionsadministered on days 1, 7 and 21; PPP=three ecdhMUC-1/ΔCtΔTm CD40Lprotein subcutaneous injections administered on days 1, 7 and 21; orVPP=a single Ad-sig-ecdhMUC-1/ΔCtΔTm CD40L vector subcutaneous injectionfollowed at days 7 and 21 by ecdhMUC-1/ΔCtΔTm CD40L protein subcutaneousinjections. See FIG. 7 for further details. The mice were challenged oneweek later with a subcutaneous injection of five hundred thousandLL2/LL1hMUC-1 lung cancer cells, and two weeks later with an intravenousinjection of 500,000 LL2/LL1hMUC-1 tumor cells. The size of thesubcutaneous tumor nodules at day were measured by caliper at multipletime points to determine the effect of the various vaccine schedules onthe growth of the LL2/LL1hMUC-1 cells as subcutaneous nodules. Themetasteses were measured by total lung weight following sacrifice.

FIG. 7 shows that three injections of the fusion protein (PPP) without apreceding Ad-sig-ecdhMUC-1/ecdCD40L vector injection failed to inducecomplete resistance to the development of the subcutaneous LL2/LL1hMUC-1tumor. In contrast, the schedule of three successive vector injections(VVV) or one vector injection followed by two protein injections (VPP)completely suppressed the appearance of the subcutaneous LL2/LL1hMUC-1tumor.

The levels of hMUC-1 specific antibodies in these mice at 63 daysfollowing the start of the vaccination were measured (FIG. 8). Theschedule of a single vector injection followed by two successive fusionprotein boosts (VPP) induced the highest levels of hMUC-1 specificantibodies, schedule VVV was intermediate, and schedule VPP wasvirtually ineffective. Thus, cancer therapy in these animals relatedsomewhat inversely to the antibody response.

A tumor treatment (post establishment) protocol was also evaluated. Inthis schedule, subcutaneous tumor (500,000 of the LL2/LL1hMUC-1) wasadministered on day 1. The three schedules (PPP, VPP and VVV) wereaccomplished on days 5, 12 and 26. Tumor was administered i.v. on day 35and tumor development (subcutaneous and lung) evaluated at day 49.Further details are found in the legend to FIG. 9.

As shown in FIG. 9, the combination of one vector injection followed bytwo protein injections (VPP) completely suppressed the growth ofestablished subcutaneous hMUC-1 positive cancer cell tumor. Threesuccessive vector administrations (VVV) had a small therapeutic affectwhile three successive protein injections (PPP) had little to no effect.

The growth of metastatic lung nodules in the pretreatment andpost-treatment (pre-establishment) cancer models is shown in FIG. 10.The pretreatment results in FIG. 10, left hand panel show that threesuccessive fusion protein injections (PPP) did not appear to suppresslung nodule growth. In contrast, schedule VVV and schedule VPP appearedto completely suppress the engraftment of the lung cancer in the lungsof the vaccinated animals.

The post treatment results in FIG. 10, right hand panel show that thecombination of one vector injection followed by two protein injections(VPP) completely suppressed the growth of established lung nodules ofthe hMUC-1 positive cancer cells. In contrast, three successive vectoradministrations (VVV) and three successive protein injections (PPP)showed some therapeutic effect but less than for the VPP protocol.

These results suggest that the best overall cancer therapy schedule isthe VPP schedule, involving a single injection ofAd-sig-ecdhMUC-1/ecdCD40L vector followed in one week by two successivesubcutaneous injections, spaced two weeks apart, of theecdhMUC-1/ecdCD40L protein. This protocol is characterized by inductionof antibody (humoral immunity) and T cell immunity (cellular immunity)to the mucin antigen.

Boosting with ecdMUC-1/ecdCD40L soluble protein versus other solubleproteins following a primary administration of the adenoviral expressionvector encoding the same protein was evaluated in hMUC-1.Tg animalschallenged with MUC-1 expressing tumor (LL2/LL1hMUC-1 cell line).Animals were boosted with a bacterial extract containingecdMUC-1/ecdCD40 (from a bacterial host strain infected withAd-sig-ecdMUC-1/ecdCD40L vector); ecdMUC-1 linked to the keyhole limpethemocyaninin (KLH), with or without incomplete Freund's adjuvant; PBS;and control bacterial extract (from a bacterial host strain not infectedwith Ad-sig-ecdMUC-1/ecdCD40L vector). The tumor cells were given 7 daysfollowing the completion of the 2nd protein boost. The results shown inFIG. 11 indicate that boosting with ecdMUC-1/ecdCD40L soluble proteinwas superior to all other approaches.

6. Construction of Adenoviral Vectors Encoding HPV E7-CD40 Ligand FusionProtein.

Methods of generating immunity by administering and adenoviral vectorexpressing a transcription unit fusion protein constituting E7 linked toa secretable form of CD40 ligand was recently reported. Ziang et al.,“An adenoviral vector cancer vaccine that delivers a tumor-associatedantigen/CD40-ligand fusion protein to dendritic cells” Proc. Natl. Acad.Sci (USA) published Nov. 25, 2003, 10.1073/pnas.2135379100 (vol.100(25):15101).

The transcription unit included DNA encoding the signal peptide from theHGH gene upstream of DNA encoding the full length HPV type 16 E7 proteinupstream of ΔCtΔTmCD40L. DNA encoding the human growth hormone signalsequence MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQID NO: 32) was prepared by annealing phosphorylated oligonucleotides(SEQ ID NOs:33 and 34) to generate the full 26 amino acid HGH sequencewith Bgl II and Notl overhangs.

Growth hormone signal upper strand (coding sequence in italics):(SEQ ID NO: 33) 5′-GATCT CCACC ATG GCT ACA GGC TCC CGG ACG TCC CTGCTC CTG GCT TTT GGC CTG CTC TGC CTG CCC TGG CTTCAA GAG GGC AGT GCC GGC-3′ Growth hormone signal lower strand:(SEQ ID NO: 34) 3′-A GGTGG TAC CGA TGT CCG AGG GCC TGC AGG GAC GAGGAC CGA AAA CCG GAC GAG ACG GAC GGG ACC GAA GTTCTC CCG TCA CGG CCGCCGG-5′. 

Synthetic HGH signal sequence was prepared by annealing the above upperand lower strand oligos. The oligos were dissolved in 50 μl H₂O (about 3mg/ml). 1 μl from each oligo (upper and lower strand) was added to 48 μlannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and2 mM Mg-acetate) incubated at 4 minutes at 95° C., 10 minutes at 70° C.and slowly cooled to about 4° C. The annealed DNA was phosphorylatedusing T4 PNK (polynucleotide kinase) under standard conditions.

The HGH signal sequence with Bgl II and Not I overhangs was inserted viaBgl II and Not I into pShuttle-E7-ΔCtΔTmCD40L(no signal sequence) toyield pshuttle-HGH/E7-ΔCtΔTmCD40L. pShuttle-E7-ΔCtΔTmCD40L (no signalsequence) was prepared by inserting HPV-16 E7 upstream of the CD40ligand sequence as follows: Sequence encoding the full HPV-16 E7 proteinwas obtained by PCR amplifying from the HPV viral genome using thefollowing primers:

HPV 16 E 7 forward primer (SEQ ID NO: 35) 5′-ATTT  GCGGCCGC TGTAATCATGCATGGAGA-3′ HPV E7 reverse primer (SEQ ID NO: 36) 5-CC CTCGAG  TTATGGTTTCTGAGAACAGAT-3′

The resulting amplicon was HPV 16 E 7 encoding DNA with 5′ end Not I and3′ end Xho 1 restriction sites. The E7 DNA was inserted into thepShuttleΔCtΔTmCD40L between the CMV promoter and directly 5′ to thespacer of the ΔCtΔTMCD40L sequence using Not I (GCGGCCGC) and Xho I(CTCGAG). The plasmid is designated pShuttle-E7-ΔCtΔTmCD40L (no signalsequence) and was used for insertion of the HGH signal sequence upstreamof E7 to generate HGH/E7-ΔCtΔTmCD40L as already described. Thus, thetranscription unit HGH/E7-ΔCtΔTmCD40L encodes the HGH secretory signalfollowed by the full length HPV type 16 E7 followed by a 10 amino acidlinker with (FENDAQAPKS; SEQ ID NO: 37) followed by murine CD40 ligandresidues 52-260.

A transcription unit that included DNA encoding the signal sequence ofthe mouse IgG kappa chain gene upstream of DNA encoding the full lengthHPV type 16 E7 protein (“K/E7”) was generated by PCR using HPV16 plasmidand the following primers:

(primer 1)  (SEQ ID NO: 38)5′-ACG ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG-3′ (primer 2) (SEQ ID NO: 39) 5′-TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3′(primer 3)  (SEQ ID NO: 40)5′-TG CTC TGG GTT CCA GGT TCC ACT GGT GAC ATG CAT G-3′; (primer 4) (SEQ ID NO: 41) 5′-TGG GTT CCA GGT TCC ACT GGT GAC ATG CAT GGA GAT ACA CCT AC-3′;  and (primer 5)  (SEQ ID NO: 42) 5′-CCG  CTC GAG TGG TTT CTG AGA ACA GAT GGG GCA C-3.′

K/E7 with the upstream kappa signal sequence was generated by fourrounds of PCR amplification (1^(st) round: primers 4 +5; 2^(nd) round:add primer 3; 3^(rd) round: add primer 2; 4^(th) round: add primer 1).The K/E7 encoding DNA was cloned into the pcDNA™ 3.1 TOPO vector(Invitrogen, San Diego, Calif.) forming pcDNA-K/E7.

A DNA fragment that contained the mouse CD40 ligand from which thetransmembrane and cytoplasmic domain had been deleted (ΔCtΔTmCD40L) wasgenerated from a mouse CD40 ligand cDNA Plasmid (pDC406-mCD40L; ATCC)using the following PCR primers:

(SEQ ID NO: 43) 5′-CCG  CTCGAG  AAC GAC GCA CAA GCA CCA AAA AGC AAGGTC GAA GAG GAA GTA AAC CTT C-3′;  and (SEQ ID NO: 44)5′-CGCGCCGCGCGCTAG 

 GAGTTTGAGTAAGCCAAAAGATG AG-3′ (high fidelity PCR kit, Roche).Fragment ΔCtΔTmCD40L was digested with Xba I and XhoI restrictionendonucleases and then ligated into pcDNA-E7. K/E7-ΔCtΔTmCD40L fragmentwas cut from the pcDNA vector and inserted into the pShuttle plasmidusing Hind III and Xba I sites (pShuttle K/E7-CtATmCD40L). Thus, theK/E7-ΔCtΔTmCD40L fragment includes the kappa chain secretory signalfollowed by the full length HPV type 16 E7 followed by a 10 amino acidlinker (LQNDAQAPKS; SEQ ID NO: 52) followed by murine CD40 ligandresidues 52-260.

A vector encoding E7 fused to human CD40 ligand lacking a transmembranedomain is prepared by inserting “space+ΔCtΔTmCD40L(human)” (prepared asdescribed above) into the plasmid pShuttle-CMV (13) after restrictionendonuclease digestion with Hind III (AAGCTT) and Xho I (CTCGAG). Thisvector is designated pShuttleΔCtΔTmCD40L(human). Modification ofpShuttleΔCtΔTmCD40L(human) to include the HPV-16 E7 upstream of thehuman CD40 ligand sequence was accomplished essentially as describedabove for the murine CD40 ligand encoding vectors. The resulting plasmidis designated pShuttle-E7-ΔCtΔTmCD40L(human)(no signal sequence) and isused for insertion of the HGH signal sequence upstream of E7 to generateHGH/E7-ΔCtΔTmCD40L(human). Thus, the transcription unitHGH/E7-ΔCtΔTmCD40L(human) encodes the HGH secretory signal followed bythe full length HPV type 16 E7 followed by a 10 amino acid linker(FENDAQAPKS; SEQ ID NO:37) followed by human CD40 ligand residues47-261.

7. Construction of Adenoviral Vectors Encoding ratHER2(Neu)/CD40L

The overexpression of the Her-2-Neu (H2N) growth factor receptor in 30%of breast cancers is associated with increased frequency of recurrenceafter surgery, and shortened survival. Mice transgenic for the ratequivalent of HER2 (“H2N” or “rH2N”) gene and therefore tolerant of thisgene (Muller et al., Cell 54: 105-115, (1998); Gut et al. Proc. Natl.Acad. Sci. USA 89: 10578-10582, (1992)) were used as experimental hostsfor evaluating immunity in the Ad-sig-rH2N/ecdCD40L vector. In thismodel, the mouse is made transgenic for a normal unactivated ratHer-2-Neu gene under the control of a mammary specific transcriptionalpromoter such as the MMTV promoter. The MMTV promoter producesoverexpression of a non-mutant rat Her-2-Neu receptor, which isanalogous to what occurs in human breast cancer. This model producespalpable tumor nodules in the primary tissue (the breast) at 24 weeks aswell as pulmonary metastases at 32 weeks. The development of breastcancer occurs spontaneously. The cancer begins focally as a clonal eventin the breast epithelial tissue through a step-wise process (Id.).Dysplasia can be detected by 12 weeks of birth. Palpable tumors in themammary glands can be detected at 25 weeks, and metastatic breast cancerin the lung can be demonstrated in 70% of mice by 32 weeks (Id.).

Ad-sig-rH2N/ecdCD40L vector was subcutaneously administered totransgenic animals one or two times at 7 day intervals to test if animmune response could be induced against the rat Her-2-Neu antigen. Twosubcutaneous injections of the Ad-sig-rH2N/ecdCD40L vector inducedcomplete resistance to the growth of the N202 (rH2N positive) mousebreast cancer cell line, whereas one subcutaneous injection of the samevector did not induce sufficient immune response to completely suppressthe growth of the rH2N positive N202 cell line. ELISPOT assays showedthat the administration of two subcutaneous injections of theAd-sig-rH2N/ecdCD40L vector 7 days apart induced levels of rH2N specificT cells in the spleens of vaccinated mice which were 10 times higherthan the levels of rH2N specific T cells induced in mice following oneinjection of the Ad-sig-rH2N/ecdCD40L vector. Finally, the immuneresistance induced against the NT2 cells by the Ad-sig-rH2N/ecdCD40Lvector prime vaccination was better than the response obtained intransgenic animals vaccinated with irradiated cytokine positive tumorcells (mitomycin treated NM cells which had been transfected with aGMCSF transcription unit).

The rH2N specific antibody levels were also measured in mice vaccinatedwith one or two subcutaneous injections of the Ad-sig-rH2N/ecdCD40Lvector. The levels of the rH2N specific antibody levels were higherfollowing two subcutaneous injections than following a singlesubcutaneous injection of the Ad-sig-rH2N/ecdCD40L vector.

8. Construction of Adenoviral Vectors Encoding huHER2/CD40L

An adenoviral vector encoding sig ecdhuHER2/CD40L was prepared asfollows. The mouse IgG kappa chain METDTLLLWVLLLWVPGSTGD (single letteramino acid code) (SEQ ID NO: 11) was prepared by PCR amplification (SEQID NOs: 12, 13 and 45) to generate the full 21 amino acid mouse IgGkappa chain signal sequence (the start codon “ATG” is shown bolded inSEQ ID NO:12).

(SEQ ID NO: 12) 5′-CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTACTG CTG-3′ (SEQ ID NO: 13)5′-TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3′

The forward primer (SEQ ID NO: 45)5′-5′-TG CTC TGG GTT CCA GGT TCC ACT GGT GAC GAA CTC-3′The forward primer for the human HER2 extracellular domain(SEQ ID NO: 46) 5′-TCC ACT GGT GAC GAACTCACCTACCTGCCCACCAATGC-3′The reverse Primer for the human HER2 extracellular domain(SEQ ID NO: 47) 5′-GGAGCTCGAG GGCTGGGTCCCCATCAAAGCTCTC-3′sig-ecdhHER2 with the upstream kappa signal sequence is generated byfour rounds of PCR amplification (1″ round: primers SEQ ID NOs 46 and47; 2^(nd) round: primer SEQ ID NOs 45 and 47; 3^(rd) round: primer SEQID NOs 13 and 47; 4^(th) round: primer SEQ ID NOs 12 and 47). Thesig-ecdhHER2 encoding DNA can be cloned into the pcDNA™ 3.1 TOPO vector(Invitrogen, San Diego, Calif.) forming pcDNA-sig-ecdhHER2. Theadditional cloning steps described for the MUC-1/CD40 Ligand expressionvector are also applicable for the HER2/CD40 ligand expression vector.

This region HER2 extracellular domain to be fused to CD40 ligandcontains two CTL epitopes; One is an HLA-A2 peptide, K I F G S L A FL(SEQ ID NO:48) representing amino acids 369-377. This peptide elicitedshort-lived peptide-specific immunity in HER2 expressing cancerpatients. See Knutson et al., Immunization of cancer patients with aHER-2/neu, HLA-A2 peptide, Clin Cancer Res. 2002May;8(5):1014-8p369-377. The second epitope is EL T Y LP T NA S (SEQ IDNO: 49) (HER2 residues 63-71) also was useful in generating immunity toHER2 expressing tumor cells. See Wang et al. Essential roles oftumor-derived helper T cell epitopes for an effective peptide-basedtumor vaccine, Cancer Immun. 2003 Nov 21;3:16. The region of the HER2ecd also includes a B cell epitope P LHNQEVTAEDGTQRCEKCSKPC(SEQIDNO:50)(HER2 positions 316-339). SeeDakappagari et al., Chimeric multi-human epidermal growth factorreceptor-2 B cell epitope peptide vaccine mediates superior antitumorresponses, J Immunol. 2003 Apr. 15; 170(8):4242-53.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method of generating an immune response for thepurpose of treating existing tumor growth in an individual against aMUC1 tumor antigen, comprising: a) a first step of administering on dayone to the individual an effective amount of an adenoviral viralexpression vector, said vector comprising a transcription unit encodinga secretable fusion protein, said fusion protein comprising a secretorysignal sequence, a MUC1 tumor antigen and a CD40 ligand wherein saidCD40 ligand does not include a transmembrane domain and wherein saidfusion protein is oriented with the tumor antigen attached to theN-terminus of the extracellular domain of said CD40 ligand; b) a secondstep of administering a first boost one week from administration of theexpression vector, a composition that comprises an effective amount ofthe secretable fusion protein comprising the MUC 1 tumor antigen andCD40 ligand; and c) a third step of administering a second boost threeweeks from administration of the expression vector, a composition thatcomprises an effective amount of the secretable fusion proteincomprising the MUC1 tumor antigen and CD40 ligand.
 2. The methodaccording to claim 1 wherein said three steps are adapted to generatecytotoxic T cell activity to the MUC 1 tumor antigen.
 3. The methodaccording to claim 2 wherein said three steps are adapted toadditionally generate antibody responses to the MUC 1 tumor antigen. 4.The method according to claim 1 wherein said second step, the firstboost is an effective amount of said adenoviral expression vector.
 5. Amethod of generating an immune response for the purpose of preventingtumor growth in an individual against a MUC1 tumor antigen, comprising:a) a first step of administering on day one to the individual aneffective amount of an adenoviral viral expression vector, said vectorcomprising a transcription unit encoding a secretable fusion protein,said fusion protein comprising a secretory signal sequence, a MUC1 tumorantigen and a CD40 ligand wherein said CD40 ligand does not include atransmembrane domain and wherein said fusion protein is oriented withthe tumor antigen attached to the N-terminus of the extracellular domainof said CD40 ligand; and b) a second step of administering a first boostapproximately one week from administration of the expression vector, asecond effective amount of said adenoviral expression vector, and c) athird step of administering a second boost approximately three weeksfrom the administration of the expression vector, a third effectiveamount of said adenoviral expression vector.
 6. A method of generatingin an individual an increased cellular immune response against a MUC1tumor antigen, comprising: a) a first step of administering on day oneto the individual an effective amount of an adenoviral viral expressionvector, said vector comprising a transcription unit encoding asecretable fusion protein, said fusion protein comprising a secretorysignal sequence, a MUC1 tumor antigen and a CD40 ligand wherein saidCD40 ligand does not include a transmembrane domain and wherein saidfusion protein is oriented with the tumor antigen attached to theN-terminus of the extracellular domain of said CD40 ligand; and b) atleast a second step of administering at least a first boost one weekfrom administration of the expression vector, another effective amountof said adenoviral expression vector.
 7. A method of generating animmune response for the purpose of both, generating enhanced cytotoxic Tcell activity and enhanced antibody activity, in an individual, againsta MUC1 tumor antigen, comprising: a) a first step of administering onday one to the individual an effective amount of an adenoviral viralexpression vector, said vector comprising a transcription unit encodinga secretable fusion protein, said fusion protein comprising a secretorysignal sequence a MUC1 tumor antigen and a CD40 ligand wherein said CD40ligand does not include a transmembrane domain and wherein said fusionprotein is oriented with the tumor antigen attached to the N-terminus ofthe extracellular domain of said CD40 ligand; b) a second step ofadministering a first boost one week from the administration of theexpression vector a composition that comprises an effective amount of afusion protein comprising the MUC1 tumor antigen and CD40 ligand; and c)a third step of administering a second boost three weeks fromadministration of the expression vector a composition that comprises aneffective amount of the fusion protein applied via the first boost. 8.The method according to claim 7 wherein said three steps are adapted togenerate enhanced cytotoxic T cell activity against the MUC1 tumorantigen.
 9. The method according to claim 7 wherein said three steps areadapted to additionally generate enhanced antibody responses to the MUC1tumor antigen.
 10. A method of generating an immune response in anindividual for the purpose of both generating enhanced cytotoxic T cellactivity and enhanced antibody activity, against a rH2N tumor antigen,comprising: a) a first step of administering on day one to theindividual an effective amount of an adenoviral viral expression vector,said vector comprising a transcription unit encoding a secretable fusionprotein, said fusion protein comprising a secretory signal sequence arH2N tumor antigen and a CD40 ligand wherein said CD40 ligand does notinclude a transmembrane domain and wherein said fusion protein isoriented with the tumor antigen attached to the N-terminus of theextracellular domain of said CD40 ligand; and b) at least a second stepof administering one week from the administration of the expressionvector, another effective amount of said adenoviral expression vector.